James Madison University JMU Scholarly Commons Senior Honors Projects, 2010-current Honors College Spring 2017 Searching for sex differences in snake skin Sydney E. Ashton James Madison University Follow this and additional works at: https://commons.lib.jmu.edu/honors201019 Part of the Integrative Biology Commons Recommended Citation Ashton, Sydney E., "Searching for sex differences in snake skin" (2017). Senior Honors Projects, 2010-current. 337. https://commons.lib.jmu.edu/honors201019/337 This Thesis is brought to you for free and open access by the Honors College at JMU Scholarly Commons. It has been accepted for inclusion in Senior Honors Projects, 2010-current by an authorized administrator of JMU Scholarly Commons. For more information, please contact [email protected]. Searching for Sex Differences in Snake Skin _______________________ An Honors College Project Presented to the Faculty of the Undergraduate College of Science and Mathematics James Madison University _______________________ by Sydney E. Ashton May 2017 Accepted by the faculty of the Department of Biology, James Madison University, in partial fulfillment of the requirements for the Honors College. FACULTY COMMITTEE: HONORS COLLEGE APPROVAL: Project Advisor: M. Rockwell Parker, Ph.D. Bradley R. Newcomer, Ph.D., Assistant Professor, Biology Dean, Honors College Reader: Ray A. Enke, Ph.D. Assistant Professor, Biology Reader: Bisi T. Velayudhan, Ph.D. Assistant Professor, Biology PUBLIC PRESENTATION This work is accepted for presentation, in part or in full, at annual meeting of the Society for Integrative and Comparative Biology in New Orleans, LA on Jan. 6, 2017. Table of Contents List of Figures 3 Acknowledgments 4 Abstract 5 Introduction i. Sexual signals in vertebrates 6 ii. Thamnophis sirtalis parietalis and its sex pheromones 9 Materials and Methods i. Animal collection 11 ii. RNA extraction and cDNA synthesis 13 iii. Primer design 14 iv. PCR 17 Results i. Semi-quantitative RT-PCR 19 ii. Quantitative RT-PCR 21 Discussion 23 References 26 2 List of Figures Figures 1 Garter snake mass and SVL 12 2 Sample Esr-α protein alignments 15 3 Plate design for quantitative RT-PCR experiments 18 4 Semi-quantitative RT-PCR products on agarose gel 19 5 Liver expression of Esr1 and Gapdh 20 6 Relative RT-PCR band densities from ImageJ analysis 20 7 Melt curves from Esr1 and Esr2 quantitative RT-PCR experiments 22 8 Expression of Esr1 and Esr2 in female and male skin 22 Tables 1 Ethogram of red-sided garter snake courtship behavior 12 2 Number of primer pairs designed and tested for each gene of interest 15 3 Amplicon length, sequence and melting temperatures for primers used in semi-quantitative and quantitative RT-PCR experiments 15 4 Semi-quantitative and quantitative RT-PCR cycle 19 3 Acknowledgments I would first like to thank my adviser and mentor, Rocky Parker for the time, resources and funding he devoted to this project and, more importantly, my development as a scientist and leader. I’d like to thank my thesis committee, Ray Enke and Bisi Velayudhan, for their support and advice throughout this research as well as the James Madison University Honors College for providing me with the framework for academic growth and achievement in my undergraduate career. Shannon Richard helped with animal collection, courtship trials, tissue collection and RNA extraction. Shannon Richard and Ricky Flores assisted with animal care. Ray Enke and Christy Schnitzler offered advice on initial stages of the design. Thanks to the James Madison University Department of Biology for providing resources for this research and Manitoba Conservation for allowing the field component of this work, including animal collection. I’d also like to thank Dave Roberts for providing logistical support in the field and Corey Cleland for influencing me to pursue research. 4 Abstract The production of many secondary sexual signals, including pheromones, is controlled by sex hormone action at the sites of signal synthesis. The red-garter snake (Thamnophis sirtalis parietalis) is an ideal vertebrate for studying the interaction between steroids and sexual signals: males exclusively rely on skin-based female pheromones during courtship, and pheromone composition is augmented by treatment with sex steroids (e.g., males produce female pheromone if implanted with estrogen). But how do steroid hormones promote pheromone expression at the molecular level in snake skin? Feminizing effects of estrogens on sexual signals are known to result from activation of estrogen receptors α (Esr1) and/or β (Esr2), while masculinizing effects of androgens arise from androgen receptor (AR) activation. We hypothesized that Esr1 and Esr2 are expressed in garter snake skin but their expression is sex-dependent with female skin expressing higher levels of Esrs. To test this, red-sided garter snakes (n=7 males, n=7 females) were collected in the spring mating season, and mRNAs from skin and control tissues (liver) were extracted and used to synthesize cDNAs. Primers were designed using the available T. sirtalis genome (NCBI) and tested in real-time PCR reactions. While both receptor types were expressed in male and female skin, Esr1 was more highly expressed in female skin. We thus attribute the feminizing effect of estrogen on pheromone phenotype in males to their lack of circulating estrogen and subsequently dormant Esrs. 5 Introduction i. Sexual signals in vertebrates Sexual dimorphism, the expression of different characteristics between males and females of a single species, is ubiquitous throughout the major groups of vertebrates, from fish to birds. Male lions (Panthera leo), for example, have large manes that signal their dominance and age whereas females do not (West and Packer, 2002). Further, sexually dimorphic features may be used as sexual signals, allowing conspecifics to communicate with one another. These signals allow individuals to recognize conspecifics and the subsequent assessment of traits such as size, sex and body condition (McLean et al., 2012). Sexual signals can be morphological, behavioral or physiological. Morphological signals include characteristics such as body size or color, whereas behavioral signals include visual displays and acoustic signals (Andersson, 1994). Female poison frogs (Dendrobatidae family), for example, can be much larger than males (Shine, 1979), and male zebra finches (Taeniopygia guttata) have brightly colored beaks compared to females (McGraw, 2006). Both would be examples of morphological signals that can promote sexually dimorphic behavioral signals, such as calling behavior or courtship dances, respectively. Further, the expression of many sexual signals is controlled by sex steroid hormones. In the case of the zebra finch, for example, the beaks of testosterone-treated females will change from dull orange to the bright red, male-typical coloration and elicit interest from females (McGraw, 2006). Similarly, chemical signals allow conspecifics to communicate with one another, often via pheromones that exhibit pronounced sexual dimorphism. 6 Pheromones, which are used to facilitate reproduction in both vertebrates and invertebrates, can exist as single compounds or, more often, as blends of chemicals that differ between the sexes. An example of pheromone signaling in vertebrates comes from birds. Mallards (Anas platyrhynchos) use pheromones in mate choice and secrete these pheromones from the uropygial gland, a sebaceous gland present in most birds, that secretes waxes primarily used in waterproofing. Uropygial pheromone production in female mallards is mediated by estrogens. Moreover, males will produce the female typical blend of pheromone compounds when injected with estrogens (van Oordt, 1931; Caro et al., 2014). Goats (Capra aegagrus) experience a phenomenon known as the “male effect” in which a primer pheromone produced by males increases luteinizing hormone pulse frequency and induces estrus in females; females treated with testosterone will produce the primer pheromone and experience the characteristics associated with the male effect (Kakuma et al., 2007). Similarly, castrated geckos (Goniurosaurus lichtenfelderi) show decreased expression of male chemical cues, which returns to the male typical composition when castrated males are supplemented with testosterone (Golinski et al., 2015). Thus, sexual signals are not only sexually dimorphic in their expression, but their expression is typically mediated by sex steroid hormones. Due to their lipid-soluble nature, sex steroid hormones, more specifically estrogens and androgens, cross the plasma membrane to enter the cell where they bind to specific receptors to cause hormone action. In the canonical pathway, estrogen receptors α and β (genes Esr1, and Esr2) and androgen receptor (AR) act as nuclear receptors that are activated by the binding of their cognate ligands, estrogens and 7 androgens, respectively. Activated nuclear receptors function primarily as transcription factors to control gene expression. Sex steroid hormones therefore affect target tissues in which their specific receptors are expressed. Generally, sex steroid hormones primarily target the brain and multiple reproductive tissues. In the green anole lizard (Anolis carolinensis), for example, AR is expressed in areas of the brain associated with reproductive behaviors (Rosen et al., 2002). One characteristic of the male copulatory system is the presence of two hemipenes (intromittent organs), expression of which is present in both embryonic males and